Fitness costs restrict niche expansion by generalist niche-constructing pathogens

Abstract

We investigated the molecular and ecological mechanisms involved in niche expansion, or generalism, versus specialization in sympatric plant pathogens. Nopaline-type and octopine-type Agrobacterium tumefaciens engineer distinct niches in their plant hosts that provide different nutrients: nopaline or octopine, respectively. Previous studies revealed that nopaline-type pathogens may expand their niche to also assimilate octopine in the presence of nopaline, but consequences of this phenomenon on pathogen dynamics in planta were not known. Here, we provided molecular insight into how the transport protein NocT can bind octopine as well as nopaline, contributing to niche expansion. We further showed that despite the ability for niche expansion, nopaline-type pathogens had no competitive advantage over octopine-type pathogens in co-infected plants. We also demonstrated that a single nucleotide polymorphism in the nocR gene was sufficient to allow octopine assimilation by nopaline-type strains even in absence of nopaline. The evolved nocR bacteria had higher fitness than their ancestor in octopine-rich transgenic plants but lower fitness in tumors induced by octopine-type pathogens. Overall, this work elucidates the specialization of A. tumefaciens to particular opine niches and explains why generalists do not always spread despite the advantage associated with broader nutritional niches.

Figure 1

Assimilation of nopaline and octopine in A. tumefaciens C58 and A. tumefaciens R10. (a) The graph indicates the OD_600nm of 18 h A. tumefaciens cultures (C58 WT, C58 ocd mutant and R10 WT backgrounds) supplemented either with nopaline or octopine or nopaline and octopine (1 mm each) as sole sources of C and N (initial OD_600nm was 0.2). Below are shown the concentrations of nopaline and octopine remaining in the cultures after 18 h. (b) Important molecular actors for the opine-niche construction (Nos and Ocs), transport (NocT and OccJ), assimilation (NoxAB and OoxAB) and gene regulation (NocR and OccR) in A. tumefaciens C58 and R10 are represented. The opine niches are constructed in the plant tumor cells through the expression of the opine synthase genes nos and ocs encoded by the T-DNA. Then, the opine niches are exploited when nopaline and octopine are imported and assimilated by A. tumefaciens that colonize the plant tumor.

Figure 2

NocT binding site. (a) octopine bound to the ligand binding site of NocT (residues from lobe 1 are in gray, whereas residues from lobe 2 are in blue) is shown as pink/magenta for the arginine/pyruvate part, respectively, in its simulated annealing Fo-Fc omit map contoured at 4 σ. Hydrogen bonds between octopine and NocT are shown as dashed lines in black for distance below 3.2 Å and in magenta for distances between 3.2 Å and 3.4 Å. (b) NocT-octopine versus NocT-nopaline. Structural comparison between the binding sites of NocT in complex with octopine (shown as pink/magenta stick) and nopaline (shown as pink/lime green stick).

Figure 3

Fluorescence and ITC K_D measurements. (a) wild-type NocT fluorescence monitoring on titration with octopine and fit (solid line) to a single binding model using Origin. Measures were done in triplicates. N/A: non acquired. (b) ITC measurements: The top panels show heat differences on injection of octopine with WT Noct and M117N mutant and lower panels show integrated heats of injection and the best fit (solid line) to a single binding model using Microcal Origin. Fitting values are indicated above.

Figure 4

Characteristics of the derived octopine-users. (a) Growth of the ancestral strain and several derived strains on octopine and nopaline (3 mm) as sole sources of carbon and nitrogen. OD_600nm after 24 h in culture are shown (initial OD_600nm=0.05). On the right are indicated the nocR mutations carried by each variant. (b) Physical representation of the noc genes (left) and expression fold changes (Fch) of the nocT and noxB genes in different variants comparatively to the parental clones calculated by RT-qPCR (right). s.ds. were calculated from two independent experiments.

Figure 5

Fitness of a derived octopine-user versus its A. tumefaciens (pTi-nos::Km) parent in different plant tumors. (a) When inoculated into WT host plants, the ancestral A. tumefaciens (pTi-nos::Km) outcompeted the derived octopine-user oct+13 as shown by the significant difference in strain proportions between inoculum and tumors (Fisher's Exact test P=1.6x10⁻⁵). (b) When inoculated into OCT+ transgenic plants, however, the derived octopine-user oct+13 outcompeted its ancestor (Fisher's Exact test P=0.005). (c) In the triple inoculation including the octopine niche-constructor and user, R10, into WT host plants, the ancestral A. tumefaciens (pTi-nos::Km) outcompeted the derived octopine-user oct+13 (Fisher's Exact test P=0.04). Mean and s.d. of inoculum was calculated from 3 independent samples, of tumors, from 10, 9 and 7 tumors for a, b and c, respectively. All experiments were done on 32 days post-infection A. thaliana plant tumors. On the right of each graph are indicated the octopine concentrations of the analyzed tumors. s.ds were calculated from 3 independent tumors.